Integrated Microfluidic Platform for Studies on Membrane Proteins and Drug Screening Assays

Abstract

The combination of microfluidics with BLM (bilayer lipid membrane) experimentation provides a promising route towards high-throughput screening platforms for studies and drug screening on membrane proteins. The microfluidic format is ideal for multiplexed and automated assays, the miniaturization of the device goes together with enhanced electrical capabilities and more stable bilayers, and dual optical and electrical measurements are possible with a horizontal configuration of the membrane. In that context, we have developed a sandwich device consisting of two glass substrates separated by a Teflon membrane. The glass substrates contain two independent and orthogonal microfluidic channels being the two fluidic reservoirs for BLM experimentation; the Teflon film presents a micrometer-size aperture, located at the intersection of the microchannels, and across which BLMs are made. Leakage-free assembly of the three layers is demonstrated using an optical adhesive. The closed configuration of the device prompted us to develop a novel methodology for BLM preparation. Lipid solution and buffer are successively flushed in both channels, so that the lipid plug deposited in the aperture spontaneously thins into a bilayer. BLM formation is monitored electrically (patch-amplifier) and optically ((fluorescence) microscopy). This lipid-plug-thinning technique gives highly stable BLMs (> 7 hrs lifetime) with an almost 100% success yield. The resulting membranes exhibit reproducible characteristics in terms of sealing quality (14 ± 4 giga-ohm) and surface area (∼80% aperture coverage, measured optically and electrically (10 ± 3.4 pF capacitance)). Insertion of alpha-hemolysin (in the BLM) confirms the formation of a bilayer structure and demonstrates the potential of the platform for single protein measurements. We will present the detailed fabrication of the microfluidic platform, BLM formation and characterization in the closed environment, and on-chip single proteins studies using optical and electrical techniques.